Solid forms of 3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl) benzoic acid

ABSTRACT

The present invention relates to a substantially crystalline and free solid state form of 3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoic acid (Form I), pharmaceutical compositions thereof, and methods of treatment therewith.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.14/470,836, filed Aug. 27, 2014, which is a divisional of Ser. No.13/933,223, filed Jul. 2, 2013, now U.S. Pat. No. 8,846,718, which is adivisional of U.S. patent application Ser. No. 12/327,902, filed Dec. 4,2008, now U.S. Pat. No. 8,507,534 B2, which claims the benefit under 35U.S.C. §119 to U.S. provisional patent application Ser. No. 61/012,162,filed Dec. 7, 2007, the entire contents of all prior applications areincorporated herein by reference.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to solid state forms, for example,crystalline forms, of3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoicacid, pharmaceutical compositions thereof, and methods therewith.

BACKGROUND OF THE INVENTION

CFTR is a cAMP/ATP-mediated anion channel that is expressed in a varietyof cells types, including absorptive and secretory epithelia cells,where it regulates anion flux across the membrane, as well as theactivity of other ion channels and proteins. In epithelia cells, normalfunctioning of CFTR is critical for the maintenance of electrolytetransport throughout the body, including respiratory and digestivetissue. CFTR is composed of approximately 1480 amino acids that encode aprotein made up of a tandem repeat of transmembrane domains, eachcontaining six transmembrane helices and a nucleotide binding domain.The two transmembrane domains are linked by a large, polar, regulatory(R)-domain with multiple phosphorylation sites that regulate channelactivity and cellular trafficking.

The gene encoding CFTR has been identified and sequenced (See Gregory,R. J. et al. (1990) Nature 347:382-386; Rich, D. P. et al. (1990) Nature347:358-362), (Riordan, J. R. et al. (1989) Science 245:1066-1073). Adefect in this gene causes mutations in CFTR resulting in cysticfibrosis (“CF”), the most common fatal genetic disease in humans. Cysticfibrosis affects approximately one in every 2,500 infants in the UnitedStates. Within the general United States population, up to 10 millionpeople carry a single copy of the defective gene without apparent illeffects. In contrast, individuals with two copies of the CF associatedgene suffer from the debilitating and fatal effects of CF, includingchronic lung disease.

In patients with cystic fibrosis, mutations in CFTR endogenouslyexpressed in respiratory epithelia leads to reduced apical anionsecretion causing an imbalance in ion and fluid transport. The resultingdecrease in anion transport contributes to enhanced mucus accumulationin the lung and the accompanying microbial infections that ultimatelycause death in CF patients. In addition to respiratory disease, CFpatients typically suffer from gastrointestinal problems and pancreaticinsufficiency that, if left untreated, results in death. In addition,the majority of males with cystic fibrosis are infertile and fertilityis decreased among females with cystic fibrosis. In contrast to thesevere effects of two copies of the CF associated gene, individuals witha single copy of the CF associated gene exhibit increased resistance tocholera and to dehydration resulting from diarrhea—perhaps explainingthe relatively high frequency of the CF gene within the population.

Sequence analysis of the CFTR gene of CF chromosomes has revealed avariety of disease causing mutations (Cutting, G. R. et al. (1990)Nature 346:366-369; Dean, M. et al. (1990) Cell 61:863:870; and Kerem,B-S. et al. (1989) Science 245:1073-1080; Kerem, B-S et al. (1990) Proc.Natl. Acad. Sci. USA 87:8447-8451). To date, >1000 disease causingmutations in the CF gene have been identified(http://www.genet.sickkids.on.ca/cftr/). The most prevalent mutation isa deletion of phenylalanine at position 508 of the CFTR amino acidsequence, and is commonly referred to as ΔF508-CFTR. This mutationoccurs in approximately 70% of the cases of cystic fibrosis and isassociated with a severe disease.

The deletion of residue 508 in ΔF508-CFTR prevents the nascent proteinfrom folding correctly. This results in the inability of the mutantprotein to exit the ER, and traffic to the plasma membrane. As a result,the number of channels present in the membrane is far less than observedin cells expressing wild-type CFTR. In addition to impaired trafficking,the mutation results in defective channel gating. Together, the reducednumber of channels in the membrane and the defective gating lead toreduced anion transport across epithelia leading to defective ion andfluid transport. (Quinton, P. M. (1990), FASEB J. 4: 2709-2727). Studieshave shown, however, that the reduced numbers of ΔF508-CFTR in themembrane are functional, albeit less than wild-type CFTR. (Dalemans etal. (1991), Nature Lond. 354: 526-528; Denning et al., supra; Pasyk andFoskett (1995), J. Cell. Biochem. 270: 12347-50). In addition toΔF508-CFTR, other disease causing mutations in CFTR that result indefective trafficking, synthesis, and/or channel gating could be up- ordown-regulated to alter anion secretion and modify disease progressionand/or severity.

Although CFTR transports a variety of molecules in addition to anions,it is clear that this role (the transport of anions) represents oneelement in an important mechanism of transporting ions and water acrossthe epithelium. The other elements include the epithelial Na⁺ channel,ENaC, Na⁺/2Cl⁻/K⁺ co-transporter, Na⁺—K⁺-ATPase pump and the basolateralmembrane K⁺ channels, that are responsible for the uptake of chlorideinto the cell.

These elements work together to achieve directional transport across theepithelium via their selective expression and localization within thecell. Chloride absorption takes place by the coordinated activity ofENaC and CFTR present on the apical membrane and the Na⁺—K⁺-ATPase pumpand Cl— channels expressed on the basolateral surface of the cell.Secondary active transport of chloride from the luminal side leads tothe accumulation of intracellular chloride, which can then passivelyleave the cell via Cl⁻ channels, resulting in a vectorial transport.Arrangement of Na⁺/2Cl⁻/K⁺ co-transporter, Na⁺—K⁺-ATPase pump and thebasolateral membrane K⁺ channels on the basolateral surface and CFTR onthe luminal side coordinate the secretion of chloride via CFTR on theluminal side. Because water is probably never actively transporteditself, its flow across epithelia depends on tiny transepithelialosmotic gradients generated by the bulk flow of sodium and chloride.

As discussed above, it is believed that the deletion of residue 508 inΔF508-CFTR prevents the nascent protein from folding correctly,resulting in the inability of this mutant protein to exit the ER, andtraffic to the plasma membrane. As a result, insufficient amounts of themature protein are present at the plasma membrane and chloride transportwithin epithelial tissues is significantly reduced. Infact, thiscellular phenomenon of defective ER processing of ABC transporters bythe ER machinery, has been shown to be the underlying basis not only forCF disease, but for a wide range of other isolated and inheriteddiseases. The two ways that the ER machinery can malfunction is eitherby loss of coupling to ER export of the proteins leading to degradation,or by the ER accumulation of these defective/misfolded proteins [AridorM, et al., Nature Med., 5(7), pp 745-751 (1999); Shastry, B. S., et al.,Neurochem. International, 43, pp 1-7 (2003); Rutishauser, J., et al.,Swiss Med Wkly, 132, pp 211-222 (2002); Morello, J P et al., TIPS, 21,pp. 466-469 (2000); Bross P., et al., Human Mut., 14, pp. 186-198(1999)].

3-(6-(1-(2,2-Difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoicacid in salt form is disclosed in International PCT Publication WO2007056341 (said publication being incorporated herein by reference inits entirety) as a modulator of CFTR activity and thus useful intreating CFTR-mediated diseases such as cystic fibrosis. However, thereis a need for stable solid forms of said compound that can be usedreadily in pharmaceutical compositions suitable for use as therapeutics.

SUMMARY OF THE INVENTION

The present invention relates to solid forms of3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoicacid (hereinafter “Compound 1”) which has the structure below:

Compound 1 and pharmaceutically acceptable compositions thereof areuseful for treating or lessening the severity of cystic fibrosis. In oneaspect, Compound 1 is in a substantially crystalline and salt free formreferred to as Form I as described and characterized herein.

Processes described herein can be used to prepare the compositions ofthis invention comprising Form I. The amounts and the features of thecomponents used in the processes would be as described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an X-ray diffraction pattern calculated from a single crystalstructure of Compound 1 in Form I.

FIG. 2 is an actual X-ray powder diffraction pattern of Compound 1 inForm I.

FIG. 3 is an overlay of an X-ray diffraction pattern calculated from asingle crystal of Compound 1 in Form I, and an actual X-ray powderdiffraction pattern of Compound 1 in Form I.

FIG. 4 is a differential scanning calorimetry (DSC) trace of Compound 1in Form I.

FIG. 5 is a conformational picture of Compound 1 in Form I based onsingle crystal X-ray analysis.

FIG. 6 is a conformational picture of Compound 1 in Form I based onsingle crystal X-ray analysis as a dimer formed through the carboxylicacid groups.

FIG. 7 is a conformational picture of Compound 1 in Form I based onsingle crystal X-ray analysis showing that the molecules are stackedupon each other.

FIG. 8 is conformational picture of Compound 1 in Form I based on singlecrystal X-ray analysis showing a different view (down a).

FIG. 9 is an ¹HNMR analysis of Compound 1 in Form I in a 50 mg/mL, 0.5methyl cellulose-polysorbate 80 suspension at T(0).

FIG. 10 is an ¹HNMR analysis of Compound 1 in Form I in a 50 mg/mL, 0.5methyl cellulose-polysorbate 80 suspension stored at room temperaturefor 24 hours.

FIG. 11 is an ¹HNMR analysis of Compound 1.HCl standard.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

As used herein, the following definitions shall apply unless otherwiseindicated.

The term “CFTR” as used herein means cystic fibrosis transmembraneconductance regulator or a mutation thereof capable of regulatoractivity, including, but not limited to, ΔF508 CFTR and G551D CFTR (see,e.g., http://www.genet.sickkids.on.ca/cftr/, for CFTR mutations).

As used herein “crystalline” refers to compounds or compositions wherethe structural units are arranged in fixed geometric patterns orlattices, so that crystalline solids have rigid long range order. Thestructural units that constitute the crystal structure can be atoms,molecules, or ions. Crystalline solids show definite melting points.

The term “modulating” as used herein means increasing or decreasing,e.g. activity, by a measurable amount.

In one aspect, the invention features a form of3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoicacid characterized as Form I.

In another embodiment, Form I is characterized by one or more peaks at15.2 to 15.6 degrees, 16.1 to 16.5 degrees, and 14.3 to 14.7 degrees inan X-ray powder diffraction obtained using Cu K alpha radiation.

In another embodiment, Form I is characterized by one or more peaks at15.4, 16.3, and 14.5 degrees.

In another embodiment, Form I is further characterized by a peak at 14.6to 15.0 degrees.

In another embodiment, Form I is further characterized by a peak at 14.8degrees.

In another embodiment, Form I is further characterized by a peak at 17.6to 18.0 degrees.

In another embodiment, Form I is further characterized by a peak at 17.8degrees.

In another embodiment, Form I is further characterized by a peak at 16.4to 16.8 degrees.

In another embodiment, Form I is further characterized by a peak at 16.4to 16.8 degrees.

In another embodiment, Form I is further characterized by a peak at 16.6degrees.

In another embodiment, Form I is further characterized by a peak at 7.6to 8.0 degrees.

In another embodiment, Form I is further characterized by a peak at 7.8degrees.

In another embodiment, Form I is further characterized by a peak at 25.8to 26.2 degrees.

In another embodiment, Form I is further characterized by a peak at 26.0degrees.

In another embodiment, Form I is further characterized by a peak at 21.4to 21.8 degrees.

In another embodiment, Form I is further characterized by a peak at 21.6degrees.

In another embodiment, Form I is further characterized by a peak at 23.1to 23.5 degrees.

In another embodiment, Form I is further characterized by a peak at 23.3degrees.

In some embodiments, Form I is characterized by a diffraction patternsubstantially similar to that of FIG. 1.

In some embodiments, Form I is characterized by a diffraction patternsubstantially similar to that of FIG. 2.

In some embodiments, the particle size distribution of D90 is about 82μm or less for Form I.

In some embodiments, the particle size distribution of D50 is about 30μm or less for Form I.

In one aspect, the invention features a pharmaceutical compositioncomprising Form I and a pharmaceutically acceptable carrier.

In one aspect, the present invention features a method of treating aCFTR mediated disease in a human comprising administering to the humanan effective amount of Form I.

In some embodiments, the method comprises administering an additionaltherapeutic agent.

In some embodiments, the disease is selected from cystic fibrosis,hereditary emphysema, hereditary hemochromatosis,coagulation-fibrinolysis deficiencies, such as protein C deficiency,Type 1 hereditary angioedema, lipid processing deficiencies, such asfamilial hypercholesterolemia, Type 1 chylomicronemia,abetalipoproteinemia, lysosomal storage diseases, such as I-celldisease/pseudo-Hurler, mucopolysaccharidoses, Sandhof/Tay-Sachs,Crigler-Najjar type II, polyendocrinopathy/hyperinsulemia, Diabetesmellitus, Laron dwarfism, myleoperoxidase deficiency, primaryhypoparathyroidism, melanoma, glycanosis CDG type 1, hereditaryemphysema, congenital hyperthyroidism, osteogenesis imperfecta,hereditary hypofibrinogenemia, ACT deficiency, Diabetes insipidus (DI),neurophyseal DI, neprogenic DI, Charcot-Marie Tooth syndrome,Perlizaeus-Merzbacher disease, neurodegenerative diseases such asAlzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis,progressive supranuclear plasy, Pick's disease, several polyglutamineneurological disorders such as Huntington, spinocerebullar ataxia typeI, spinal and bulbar muscular atrophy, dentatorubal pallidoluysian, andmyotonic dystrophy, as well as spongiform encephalopathies, such ashereditary Creutzfeldt-Jakob disease, Fabry disease,Straussler-Scheinker syndrome, COPD, dry-eye disease, and Sjogren'sdisease.

In one embodiment, the present invention provides a method of treatingcystic fibrosis in a human, comprising administering to said human aneffective amount of Form I.

In one aspect, the present invention features a kit comprising Form Iand instructions for use thereof.

In one aspect, the present invention features a process of preparingForm I comprising dispersing or dissolving the HCl salt of3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoicacid in an appropriate solvent for an effective amount of time.

In one embodiment, the present invention features a process of preparingForm I comprising dispersing the HCl salt of3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoicacid in an appropriate solvent for an effective amount of time.

In some embodiments, the appropriate solvent is water or a alcohol/watermixture.

In some embodiments, the appropriate solvent is water or 50%methanol/water mixture.

In some embodiments, the appropriate solvent is water.

In some embodiments, the appropriate solvent is a mixture comprising 50%methanol and 50% water.

In some embodiments, the effective amount of time is about 2 to about aday. In some embodiments, the effective amount of time is about 2 toabout 18 hours. In some embodiments, the effective amount of time isabout 2 to about 12 hours. In some embodiments, the effective amount oftime is about 2 to about 6 hours.

In one aspect, the invention features a crystal form of3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoicacid having a monoclinic crystal system, a P2₁/n space group, and thefollowing unit cell dimensions: a=4.9626 (7) Å, b=12.2994 (18) Å,c=33.075 (4) Å, α=90°, β=93.938 (9)°, and γ=90°.

Methods of Preparing Form I.

In one embodiment, Form I is prepared from dispersing or dissolving asalt form, such as HCL, of3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoicacid in an appropriate solvent for an effective amount of time. Inanother embodiment, Form I is prepared from dispersing a salt form, suchas HCL, of3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoicacid in an appropriate solvent for an effective amount of time. Inanother embodiment, Form I is formed directly from3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)-t-butylbenzoateand an appropriate acid, such as formic acid. In one embodiment, the HClsalt form of3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoicacid is the starting point and in one embodiment can be prepared bycoupling an acid chloride moiety with an amine moiety according toSchemes 1-3.

Using the HCl, for example, salt form of3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoicacid as a starting point, Form I can be formed in high yields bydispersing or dissolving the HCl salt form of3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoicacid in an appropriate solvent for an effective amount of time. Othersalt forms of3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoicacid may be used such as, for example, other mineral or organic acidforms. The other salt forms result from hydrolysis of the t-butyl esterwith the corresponding acid. Other acids/salt forms include nitric,sulfuric, phosphoric, boric, acetic, benzoic, malonic, and the like. Thesalt form of3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoicacid may or may not be soluble depending upon the solvent used, but lackof solubility does not hinder formation of Form I. For example, in oneembodiment, the appropriate solvent may be water or an alcohol/watermixture such as 50% methanol/water mixture, even though the HCl saltform of3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoicacid is only sparingly soluble in water. In one embodiment, theappropriate solvent is water.

The effective amount of time for formation of Form I from the salt formof3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoicacid can be any time between 2 to 24 hours or greater. Generally,greater than 24 hours is not needed to obtain high yields (˜98%), butcertain solvents may require greater amounts of time. It is alsorecognized that the amount of time needed is inversely proportional tothe temperature. That is, the higher the temperature the less timeneeded to affect dissociation of acid to form Form I. When the solventis water, stirring the dispersion for approximately 24 hours at roomtemperature gives Form I in an approximately 98% yield. If a solution ofthe salt form of3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoicacid is desired for process purposes, an elevated temperature may beused. After stirring the solution for an effective amount of time at theelevated temperature, recrystallization upon cooling yieldssubstantially pure forms of Form I. In one embodiment, substantiallypure refers to greater than about 90% purity. In another embodiment,substantially pure refers to greater than about 95% purity. In anotherembodiment, substantially pure refers to greater than about 98% purity.In another embodiment, substantially pure refers to greater than about99% purity. The temperature selected depends in part on the solvent usedand is well within the capabilities of someone of ordinary skill in theart to determine. In one embodiment, the temperature is between roomtemperature and about 80° C. In another embodiment, the temperature isbetween room temperature and about 40° C. In another embodiment, thetemperature is between about 40° C. and about 60° C. In anotherembodiment, the temperature is between about 60° C. and about 80° C.

In some embodiments, Form I may be further purified by recrystallizationfrom an organic solvent. Examples of organic solvents include, but arenot limited to, toluene, cumene, anisole, 1-butanol, isopropylacetate,butyl acetate, isobutyl acetate, methyl t-butyl ether, methyl isobutylketone, or 1-propanol/water (at various ratios). Temperature may be usedas described above. For example, in one embodiment, Form I is dissolvedin 1-butanol at 75° C. until it is completely dissolved. Cooling downthe solution to 10° C. at a rate of 0.2° C./min yields crystals of FormI which may be isolated by filtration.

Uses, Formulation and Administration

Pharmaceutically Acceptable Compositions

In another aspect of the present invention, pharmaceutically acceptablecompositions are provided, wherein these compositions comprise Form I asdescribed herein, and optionally comprise a pharmaceutically acceptablecarrier, adjuvant or vehicle. In certain embodiments, these compositionsoptionally further comprise one or more additional therapeutic agents.

As described above, the pharmaceutically acceptable compositions of thepresent invention additionally comprise a pharmaceutically acceptablecarrier, adjuvant, or vehicle, which, as used herein, includes any andall solvents, diluents, or other liquid vehicle, dispersion orsuspension aids, surface active agents, isotonic agents, thickening oremulsifying agents, preservatives, solid binders, lubricants and thelike, as suited to the particular dosage form desired. Remington'sPharmaceutical Sciences, Sixteenth Edition, E. W. Martin (MackPublishing Co., Easton, Pa., 1980) discloses various carriers used informulating pharmaceutically acceptable compositions and knowntechniques for the preparation thereof. Except insofar as anyconventional carrier medium is incompatible with the compounds of theinvention, such as by producing any undesirable biological effect orotherwise interacting in a deleterious manner with any othercomponent(s) of the pharmaceutically acceptable composition, its use iscontemplated to be within the scope of this invention. Some examples ofmaterials which can serve as pharmaceutically acceptable carriersinclude, but are not limited to, ion exchangers, alumina, aluminumstearate, lecithin, serum proteins, such as human serum albumin, buffersubstances such as phosphates, glycine, sorbic acid, or potassiumsorbate, partial glyceride mixtures of saturated vegetable fatty acids,water, salts or electrolytes, such as protamine sulfate, disodiumhydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zincsalts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone,polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, woolfat, sugars such as lactose, glucose and sucrose; starches such as cornstarch and potato starch; cellulose and its derivatives such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; powderedtragacanth; malt; gelatin; talc; excipients such as cocoa butter andsuppository waxes; oils such as peanut oil, cottonseed oil; saffloweroil; sesame oil; olive oil; corn oil and soybean oil; glycols; such apropylene glycol or polyethylene glycol; esters such as ethyl oleate andethyl laurate; agar; buffering agents such as magnesium hydroxide andaluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline;Ringer's solution; ethyl alcohol, and phosphate buffer solutions, aswell as other non-toxic compatible lubricants such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releasingagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the composition,according to the judgment of the formulator.

Uses of Compounds and Pharmaceutically Acceptable Compositions

In yet another aspect, the present invention provides a method oftreating a condition, disease, or disorder implicated by CFTR. Incertain embodiments, the present invention provides a method of treatinga condition, disease, or disorder implicated by a deficiency of CFTRactivity, the method comprising administering a composition comprising asolid state form of Form I described herein to a subject, preferably amammal, in need thereof.

A “CFTR-mediated disease” as used herein is a disease selected fromcystic fibrosis, Hereditary emphysema, Hereditary hemochromatosis,Coagulation-Fibrinolysis deficiencies, such as Protein C deficiency,Type 1 hereditary angioedema, Lipid processing deficiencies, such asFamilial hypercholesterolemia, Type 1 chylomicronemia,Abetalipoproteinemia, Lysosomal storage diseases, such as I-celldisease/Pseudo-Hurler, Mucopolysaccharidoses, Sandhof/Tay-Sachs,Crigler-Najjar type II, Polyendocrinopathy/Hyperinsulemia, Diabetesmellitus, Laron dwarfism, Myleoperoxidase deficiency, Primaryhypoparathyroidism, Melanoma, Glycanosis CDG type 1, Hereditaryemphysema, Congenital hyperthyroidism, Osteogenesis imperfecta,Hereditary hypofibrinogenemia, ACT deficiency, Diabetes insipidus (DI),Neurophyseal DI, Neprogenic DI, Charcot-Marie Tooth syndrome,Perlizaeus-Merzbacher disease, neurodegenerative diseases such asAlzheimer's disease, Parkinson's disease, Amyotrophic lateral sclerosis,Progressive supranuclear plasy, Pick's disease, several polyglutamineneurological disorders such as Huntington, Spinocerebullar ataxia typeI, Spinal and bulbar muscular atrophy, Dentatorubal pallidoluysian, andMyotonic dystrophy, as well as Spongiform encephalopathies, such asHereditary Creutzfeldt-Jakob disease, Fabry disease,Straussler-Scheinker syndrome, COPD, dry-eye disease, and Sjogren'sdisease.

In certain embodiments, the present invention provides a method oftreating a CFTR-mediated disease in a human comprising the step ofadministering to said human an effective amount of a compositioncomprising Form I described herein.

According to an alternative preferred embodiment, the present inventionprovides a method of treating cystic fibrosis in a human comprising thestep of administering to said human a composition comprising Form Idescribed herein.

According to the invention an “effective amount” of Form I or apharmaceutically acceptable composition thereof is that amount effectivefor treating or lessening the severity of any of the diseases recitedabove.

Form I or a pharmaceutically acceptable composition thereof may beadministered using any amount and any route of administration effectivefor treating or lessening the severity of one or more of the diseasesrecited above.

In certain embodiments, Form I described herein or a pharmaceuticallyacceptable composition thereof is useful for treating or lessening theseverity of cystic fibrosis in patients who exhibit residual CFTRactivity in the apical membrane of respiratory and non-respiratoryepithelia. The presence of residual CFTR activity at the epithelialsurface can be readily detected using methods known in the art, e.g.,standard electrophysiological, biochemical, or histochemical techniques.Such methods identify CFTR activity using in vivo or ex vivoelectrophysiological techniques, measurement of sweat or salivaryCl-concentrations, or ex vivo biochemical or histochemical techniques tomonitor cell surface density. Using such methods, residual CFTR activitycan be readily detected in patients heterozygous or homozygous for avariety of different mutations, including patients homozygous orheterozygous for the most common mutation, ΔF508.

In one embodiment, Form I described herein or a pharmaceuticallyacceptable composition thereof is useful for treating or lessening theseverity of cystic fibrosis in patients within certain genotypesexhibiting residual CFTR activity, e.g., class III mutations (impairedregulation or gating), class IV mutations (altered conductance), orclass V mutations (reduced synthesis) (Lee R. Choo-Kang, Pamela L.,Zeitlin, Type I, II, III, IV, and V cystic fibrosis TansmembraneConductance Regulator Defects and Opportunities of Therapy; CurrentOpinion in Pulmonary Medicine 6:521-529, 2000). Other patient genotypesthat exhibit residual CFTR activity include patients homozygous for oneof these classes or heterozygous with any other class of mutations,including class I mutations, class II mutations, or a mutation thatlacks classification.

In one embodiment, Form I described herein or a pharmaceuticallyacceptable composition thereof is useful for treating or lessening theseverity of cystic fibrosis in patients within certain clinicalphenotypes, e.g., a moderate to mild clinical phenotype that typicallycorrelates with the amount of residual CFTR activity in the apicalmembrane of epithelia. Such phenotypes include patients exhibitingpancreatic insufficiency or patients diagnosed with idiopathicpancreatitis and congenital bilateral absence of the vas deferens, ormild lung disease.

The exact amount required will vary from subject to subject, dependingon the species, age, and general condition of the subject, the severityof the infection, the particular agent, its mode of administration, andthe like. The compounds of the invention are preferably formulated indosage unit form for ease of administration and uniformity of dosage.The expression “dosage unit form” as used herein refers to a physicallydiscrete unit of agent appropriate for the patient to be treated. Itwill be understood, however, that the total daily usage of the compoundsand compositions of the present invention will be decided by theattending physician within the scope of sound medical judgment. Thespecific effective dose level for any particular patient or organismwill depend upon a variety of factors including the disorder beingtreated and the severity of the disorder; the activity of the specificcompound employed; the specific composition employed; the age, bodyweight, general health, sex and diet of the patient; the time ofadministration, route of administration, and rate of excretion of thespecific compound employed; the duration of the treatment; drugs used incombination or coincidental with the specific compound employed, andlike factors well known in the medical arts. The term “patient”, as usedherein, means an animal, preferably a mammal, and most preferably ahuman.

The pharmaceutically acceptable compositions of this invention can beadministered to humans and other animals orally, rectally, parenterally,intracisternally, intravaginally, intraperitoneally, topically (as bypowders, ointments, or drops), bucally, as an oral or nasal spray, orthe like, depending on the severity of the infection being treated. Incertain embodiments, the compounds of the invention may be administeredorally or parenterally at dosage levels of about 0.01 mg/kg to about 50mg/kg and preferably from about 1 mg/kg to about 25 mg/kg, of subjectbody weight per day, one or more times a day, to obtain the desiredtherapeutic effect.

In certain embodiments, the dosage amount of Form I in the dosage unitform is from 100 mg to 1,000 mg. In another embodiment, the dosageamount of Form I is from 200 mg to 900 mg. In another embodiment, thedosage amount of Form I is from 300 mg to 800 mg. In another embodiment,the dosage amount of Form I is from 400 mg to 700 mg. In anotherembodiment, the dosage amount of Form I is from 500 mg to 600 mg.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables.

The injectable formulations can be sterilized, for example, byfiltration through a bacterial-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

Compositions for rectal or vaginal administration are preferablysuppositories which can be prepared by mixing the compounds of thisinvention with suitable non-irritating excipients or carriers such ascocoa butter, polyethylene glycol or a suppository wax which are solidat ambient temperature but liquid at body temperature and therefore meltin the rectum or vaginal cavity and release the active compound.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activecompound is mixed with at least one inert, pharmaceutically acceptableexcipient or carrier such as sodium citrate or dicalcium phosphateand/or a) fillers or extenders such as starches, lactose, sucrose,glucose, mannitol, and silicic acid, b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia, c) humectants such as glycerol, d) disintegratingagents such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate, e) solutionretarding agents such as paraffin, f) absorption accelerators such asquaternary ammonium compounds, g) wetting agents such as, for example,cetyl alcohol and glycerol monostearate, h) absorbents such as kaolinand bentonite clay, and i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof. In the case of capsules, tablets and pills, thedosage form may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like. The solid dosage forms of tablets, dragees, capsules, pills,and granules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally contain opacifying agents and can also be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions that can be usedinclude polymeric substances and waxes. Solid compositions of a similartype may also be employed as fillers in soft and hard-filled gelatincapsules using such excipients as lactose or milk sugar as well as highmolecular weight polyethylene glycols and the like.

The active compounds can also be in microencapsulated form with one ormore excipients as noted above. The solid dosage forms of tablets,dragees, capsules, pills, and granules can be prepared with coatings andshells such as enteric coatings, release controlling coatings and othercoatings well known in the pharmaceutical formulating art. In such soliddosage forms the active compound may be admixed with at least one inertdiluent such as sucrose, lactose or starch. Such dosage forms may alsocomprise, as is normal practice, additional substances other than inertdiluents, e.g., tableting lubricants and other tableting aids such amagnesium stearate and microcrystalline cellulose. In the case ofcapsules, tablets and pills, the dosage forms may also comprisebuffering agents. They may optionally contain opacifying agents and canalso be of a composition that they release the active ingredient(s)only, or preferentially, in a certain part of the intestinal tract,optionally, in a delayed manner. Examples of embedding compositions thatcan be used include polymeric substances and waxes.

It will also be appreciated that Form I described herein or apharmaceutically acceptable composition thereof can be employed incombination therapies, that is, Form I can be administered concurrentlywith, prior to, or subsequent to, one or more other desired therapeuticsor medical procedures. The particular combination of therapies(therapeutics or procedures) to employ in a combination regimen willtake into account compatibility of the desired therapeutics and/orprocedures and the desired therapeutic effect to be achieved. It willalso be appreciated that the therapies employed may achieve a desiredeffect for the same disorder (for example, an inventive compound may beadministered concurrently with another agent used to treat the samedisorder), or they may achieve different effects (e.g., control of anyadverse effects). As used herein, additional therapeutic agents that arenormally administered to treat or prevent a particular disease, orcondition, are known as “appropriate for the disease, or condition,being treated”.

In one embodiment, the additional agent is selected from a mucolyticagent, bronchodialator, an anti-biotic, an anti-infective agent, ananti-inflammatory agent, a CFTR modulator other than a compound of thepresent invention, or a nutritional agent.

In another embodiment, the additional agent is a compound selected fromgentamicin, curcumin, cyclophosphamide, 4-phenylbutyrate, miglustat,felodipine, nimodipine, Philoxin B, geniestein, Apigenin, cAMP/cGMPmodulators such as rolipram, sildenafil, milrinone, tadalafil, amrinone,isoproterenol, albuterol, and almeterol, deoxyspergualin, HSP 90inhibitors, HSP 70 inhibitors, proteosome inhibitors such as epoxomicin,lactacystin, etc.

In another embodiment, the additional agent is a compound disclosed inWO 2004028480, WO 2004110352, WO 2005094374, WO 2005120497, or WO2006101740.

In another embodiment, the additional agent is a benzo(c)quinoliziniumderivative that exhibits CFTR modulation activity or a benzopyranderivative that exhibits CFTR modulation activity.

In another embodiment, the additional agent is a compound disclosed inU.S. Pat. No. 7,202,262, U.S. Pat. No. 6,992,096, US20060148864,US20060148863, US20060035943, US20050164973, WO2006110483, WO2006044456,WO2006044682, WO2006044505, WO2006044503, WO2006044502, or WO2004091502.

In another embodiment, the additional agent is a compound disclosed inWO2004080972, WO2004111014, WO2005035514, WO2005049018, WO2006002421,WO2006099256, WO2006127588, or WO2007044560.

In another embodiment, the an additional agent selected from compoundsdisclosed in U.S. patent application Ser. No. 11/165,818, published asU.S. Published Patent Application No. 2006/0074075, filed Jun. 24, 2005,and hereby incorporated by reference in its entirety. In anotherembodiment, the additional agent isN-(5-hydroxy-2,4-ditert-butyl-phenyl)-4-oxo-1H-quinoline-3-carboxamide.These combinations are useful for treating the diseases described hereinincluding cystic fibrosis. These combinations are also useful in thekits described herein.

The amount of additional therapeutic agent present in the compositionsof this invention will be no more than the amount that would normally beadministered in a composition comprising that therapeutic agent as theonly active agent. Preferably the amount of additional therapeutic agentin the presently disclosed compositions will range from about 50% to100% of the amount normally present in a composition comprising thatagent as the only therapeutically active agent.

Form I described herein or a pharmaceutically acceptable compositionthereof may also be incorporated into compositions for coating animplantable medical device, such as prostheses, artificial valves,vascular grafts, stents and catheters. Accordingly, the presentinvention, in another aspect, includes a composition for coating animplantable device comprising Form I described herein or apharmaceutically acceptable composition thereof, and in classes andsubclasses herein, and a carrier suitable for coating said implantabledevice. In still another aspect, the present invention includes animplantable device coated with a composition comprising Form I describedherein or a pharmaceutically acceptable composition thereof, and acarrier suitable for coating said implantable device. Suitable coatingsand the general preparation of coated implantable devices are describedin U.S. Pat. Nos. 6,099,562; 5,886,026; and 5,304,121. The coatings aretypically biocompatible polymeric materials such as a hydrogel polymer,polymethyldisiloxane, polycaprolactone, polyethylene glycol, polylacticacid, ethylene vinyl acetate, and mixtures thereof. The coatings mayoptionally be further covered by a suitable topcoat of fluorosilicone,polysaccarides, polyethylene glycol, phospholipids or combinationsthereof to impart controlled release characteristics in the composition.

In order that the invention described herein may be more fullyunderstood, the following examples are set forth. It should beunderstood that these examples are for illustrative purposes only andare not to be construed as limiting this invention in any manner.

EXAMPLES Methods & Materials

Differential Scanning Calorimetry (DSC)

The Differential scanning calorimetry (DSC) data of Form I werecollected using a DSC Q100 V9.6 Build 290 (TA Instruments, New Castle,Del.). Temperature was calibrated with indium and heat capacity wascalibrated with sapphire. Samples of 3-6 mg were weighed into aluminumpans that were crimped using lids with 1 pin hole. The samples werescanned from 25° C. to 350° C. at a heating rate of 1.0° C./min and witha nitrogen gas purge of 50 ml/min. Data were collected by ThermalAdvantage Q Series™ version 2.2.0.248 software and analyzed by UniversalAnalysis software version 4.1D (TA Instruments, New Castle, Del.). Thereported numbers represent single analyses.

XRPD (X-Ray Powder Diffraction)

The X-Ray diffraction (XRD) data of Form 1 were collected on a Bruker D8DISCOVER powder diffractometer with HI-STAR 2-dimensional detector and aflat graphite monochromator. Cu sealed tube with Kα radiation was usedat 40 kV, 35 mA. The samples were placed on zero-background siliconwafers at 25° C. For each sample, two data frames were collected at 120seconds each at 2 different θ₂ angles: 8° and 26°. The data wereintegrated with GADDS software and merged with DIFFRACT^(plus)EVAsoftware. Uncertainties for the reported peak positions are ±0.2degrees.

Vitride® (sodium bis(2-methoxyethoxy)aluminum hydride [orNaAlH₂(OCH₂CH₂OCH₃)₂], 65 wgt % solution in toluene) was purchased fromAldrich Chemicals.

2,2-Difluoro-1,3-benzodioxole-5-carboxylic acid was purchased fromSaltigo (an affiliate of the Lanxess Corporation).

Anywhere in the present application where a name of a compound may notcorrectly describe the structure of the compound, the structuresupersedes the name and governs.

Synthesis of3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoicacid.HCl

Acid Chloride Moiety

Commercially available 2,2-difluoro-1,3-benzodioxole-5-carboxylic acid(1.0 eq) is slurried in toluene (10 vol). Vitride® (2 eq) is added viaaddition funnel at a rate to maintain the temperature at 15-25° C. Atthe end of addition the temperature is increased to 40° C. for 2 h then10% (w/w) aq. NaOH (4.0 eq) is carefully added via addition funnelmaintaining the temperature at 40-50° C. After stirring for anadditional 30 minutes, the layers are allowed to separate at 40° C. Theorganic phase is cooled to 20° C. then washed with water (2×1.5 vol),dried (Na₂SO₄), filtered, and concentrated to afford crude(2,2-difluoro-1,3-benzodioxol-5-yl)-methanol that is used directly inthe next step.

(2,2-difluoro-1,3-benzodioxol-5-yl)-methanol (1.0 eq) is dissolved inMTBE (5 vol). A catalytic amount of DMAP (1 mol %) is added and SOCl₂(1.2 eq) is added via addition funnel. The SOCl₂ is added at a rate tomaintain the temperature in the reactor at 15-25° C. The temperature isincreased to 30° C. for 1 hour then cooled to 20° C. then water (4 vol)is added via addition funnel maintaining the temperature at less than30° C. After stirring for an additional 30 minutes, the layers areallowed to separate. The organic layer is stirred and 10% (w/v) aq. NaOH(4.4 vol) is added. After stirring for 15 to 20 minutes, the layers areallowed to separate. The organic phase is then dried (Na₂SO₄), filtered,and concentrated to afford crude5-chloromethyl-2,2-difluoro-1,3-benzodioxole that is used directly inthe next step.

A solution of 5-chloromethyl-2,2-difluoro-1,3-benzodioxole (1 eq) inDMSO (1.25 vol) is added to a slurry of NaCN (1.4 eq) in DMSO (3 vol)maintaining the temperature between 30-40° C. The mixture is stirred for1 hour then water (6 vol) is added followed by MTBE (4 vol). Afterstirring for 30 min, the layers are separated. The aqueous layer isextracted with MTBE (1.8 vol). The combined organic layers are washedwith water (1.8 vol), dried (Na₂SO₄), filtered, and concentrated toafford crude (2,2-difluoro-1,3-benzodioxol-5-yl)-acetonitrile (95%) thatis used directly in the next step.

A mixture of (2,2-difluoro-1,3-benzodioxol-5-yl)-acetonitrile (1.0 eq),50 wt % aqueous KOH (5.0 eq) 1-bromo-2-chloroethane (1.5 eq), andOct₄NBr (0.02 eq) is heated at 70° C. for 1 h. The reaction mixture iscooled then worked up with MTBE and water. The organic phase is washedwith water and brine then the solvent is removed to afford(2,2-difluoro-1,3-benzodioxol-5-yl)-cyclopropanecarbonitrile.

(2,2-difluoro-1,3-benzodioxol-5-yl)-cyclopropanecarbonitrile ishydrolyzed using 6 M NaOH (8 equiv) in ethanol (5 vol) at 80° C.overnight. The mixture is cooled to room temperature and ethanol isevaporated under vacuum. The residue is taken into water and MTBE, 1 MHCl was added and the layers are separated. The MTBE layer was thentreated with dicyclohexylamine (0.97 equiv). The slurry is cooled to 0°C., filtered and washed with heptane to give the corresponding DCHAsalt. The salt is taken into MTBE and 10% citric acid and stirred untilall solids dissolve. The layers are separated and the MTBE layer waswashed with water and brine. Solvent swap to heptane followed byfiltration gives1-(2,2-difluoro-1,3-benzodioxol-5-yl)-cyclopropanecarboxylic acid afterdrying in a vacuum oven at 50° C. overnight.

1-(2,2-difluoro-1,3-benzodioxol-5-yl)-cyclopropanecarboxylic acid (1.2eq) is slurried in toluene (2.5 vol) and the mixture heated to 60° C.SOCl₂ (1.4 eq) is added via addition funnel. The toluene and SOCl₂ aredistilled from the reaction mixture after 30 minutes. Additional toluene(2.5 vol) is added and distilled again.

Amine Moiety

2-Bromo-3-methylpyridine (1.0 eq) is dissolved in toluene (12 vol).K₂CO₃ (4.8 eq) is added followed by water (3.5 vol) and the mixtureheated to 65° C. under a stream of N₂ for 1 hour.3-(t-Butoxycarbonyl)phenylboronic acid (1.05 eq) and Pd(dppf)Cl₂.CH₂Cl₂(0.015 eq) are then added and the mixture is heated to 80° C. After 2hours, the heat is turned off, water is added (3.5 vol) and the layersare allowed to separate. The organic phase is then washed with water(3.5 vol) and extracted with 10% aqueous methanesulfonic acid (2 eqMsOH, 7.7 vol). The aqueous phase is made basic with 50% aqueous NaOH (2eq) and extracted with EtOAc (8 vol). The organic layer is concentratedto afford crude tert-butyl-3-(3-methylpyridin-2-yl)benzoate (82%) thatis used directly in the next step.

tert-Butyl-3-(3-methylpyridin-2-yl)benzoate (1.0 eq) is dissolved inEtOAc (6 vol). Water (0.3 vol) is added followed by urea-hydrogenperoxide (3 eq). The phthalic anhydride (3 eq) is added portion-wise asa solid to maintain the temperature in the reactor below 45° C. Aftercompletion of phthalic anhydride addition, the mixture is heated to 45°C. After stirring for an additional 4 hours, the heat is turned off 10%w/w aqueous Na₂SO₃ (1.5 eq) is added via addition funnel. Aftercompletion of Na₂SO₃ addition, the mixture is stirred for an additional30 minutes and the layers separated. The organic layer is stirred and10% w/w aq. Na₂CO₃ (2 eq) is added. After stirring for 30 minutes, thelayers are allowed to separate. The organic phase is washed 13% w/v aqNaCl. The organic phase is then filtered and concentrated to affordcrude 2-(3-(tert-butoxycarbonyl)phenyl)-3-methylpyridine-1-oxide (95%)that is used directly in the next step.

A solution of 2-(3-(tert-butoxycarbonyl)phenyl)-3-methylpyridine-1-oxide(1 eq) and pyridine (4 eq) in MeCN (8 vol) is heated to 70° C. Asolution of methanesulfonic anhydride (1.5 eq) in MeCN (2 vol) is addedover 50 min via addition funnel maintaining the temperature at less than75° C. The mixture is stirred for an additional 0.5 hours after completeaddition. The mixture is then allowed to cool to ambient. Ethanolamine(10 eq) is added via addition funnel. After stirring for 2 hours, water(6 vol) is added and the mixture is cooled to 10° C. After stirring forNLT 3 hours, the solid is collected by filtration and washed with water(3 vol), 2:1 MeCN/water (3 vol), and MeCN (2×1.5 vol). The solid isdried to constant weight (<1% difference) in a vacuum oven at 50° C.with a slight N₂ bleed to affordtert-butyl-3-(6-amino-3-methylpyridin-2-yl)benzoate as a red-yellowsolid (53% yield).

The crude acid chloride is dissolved in toluene (2.5 vol based on acidchloride) and added via addition funnel to a mixture oftert-butyl-3-(6-amino-3-methylpyridin-2-yl)benzoate (1 eq),dimethylaminopyridine (DMAP, 0.02 eq), and triethylamine (3.0 eq) intoluene (4 vol based ontert-butyl-3-(6-amino-3-methylpyridin-2-yl)benzoate). After 2 hours,water (4 vol based ontert-butyl-3-(6-amino-3-methylpyridin-2-yl)benzoate) is added to thereaction mixture. After stirring for 30 minutes, the layers areseparated. The organic phase is then filtered and concentrated to afforda thick oil of3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)-t-butylbenzoate(quantitative crude yield). MeCN (3 vol based on crude product) is addedand distilled until crystallization occurs. Water (2 vol based on crudeproduct) is added and the mixture stirred for 2 h. The solid iscollected by filtration, washed with 1:1 (by volume) MeCN/water (2×1 volbased on crude product), and partially dried on the filter under vacuum.The solid is dried to constant weight (<1% difference) in a vacuum ovenat 60° C. with a slight N₂ bleed to afford3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)-t-butylbenzoateas a brown solid.

To a slurry of3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)-t-butylbenzoate(1.0 eq) in MeCN (3.0 vol) is added water (0.83 vol) followed byconcentrated aqueous HCl (0.83 vol). The mixture is heated to 45±5° C.After stirring for 24 to 48 hours the reaction is complete and themixture is allowed to cool to ambient. Water (1.33 vol) is added and themixture stirred. The solid is collected by filtration, washed with water(2×0.3 vol), and partially dried on the filter under vacuum. The solidis dried to constant weight (<1% difference) in a vacuum oven at 60° C.with a slight N₂ bleed to afford3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoicacid.HCl as an off-white solid.

A slurry of3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoicacid.HCl (1 eq) in water (10 vol) is stirred at ambient temperature. Asample is taken after stirring for 24 hours. The sample is filtered andthe solid washed with water (2×). The solid sample is submitted for DSCanalysis. When DSC analysis indicates complete conversion to Form I, thesolid is collected by filtration, washed with water (2×1.0 vol), andpartially dried on the filter under vacuum. The solid is dried toconstant weight (<1% difference) in a vacuum oven at 60° C. with aslight N₂ bleed to afford Form I as an off-white solid (98% yield). ¹HNMR (400 MHz, DMSO-d6) 9.14 (s, 1H), 7.99-7.93 (m, 3H), 7.80-7.78 (m,1H), 7.74-7.72 (m, 1H), 7.60-7.55 (m, 2H), 7.41-7.33 (m, 2H), 2.24 (s,3H), 1.53-1.51 (m, 2H), 1.19-1.17 (m, 2H).

To a slurry of3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoicacid.HCl (1 eq) in water (10 vol) stirred at ambient temperature isadded 50% w/w aq. NaOH (2.5 eq). The mixture is stirred for NLT 15 minor until a homogeneous solution. Concentrated HCl (4 eq) is added tocrystallize Form I. The mixture is heated to 60° C. or 90° C. if neededto reduce the level of the t-butylbenzoate ester. The mixture is heateduntil HPLC analysis indicates NMT 0.8% (AUC) t-butylbenzoate ester. Themixture is then cooled to ambient and the solid is collected byfiltration, washed with water (3×3.4 vol), and partially dried on thefilter under vacuum. The solid is dried to constant weight (<1%difference) in a vacuum oven at 60° C. with a slight N₂ bleed to affordForm I as an off-white solid (97% yield).

A solution of3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)-t-butylbenzoate(1.0 eq) in formic acid (3.0 vol) is heated to 70±10° C. The reaction iscontinued until the reaction is complete (NMT 1.0% AUC3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)-t-butylbenzoate)or heating for NMT 8 h. The mixture is allowed to cool to ambient. Thesolution is added to water (6 vol) heated at 50° C. and the mixturestirred. The mixture is then heated to 70±10° C. until the level of3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)-t-butylbenzoateis NMT 0.8% (AUC). The solid is collected by filtration, washed withwater (2×3 vol), and partially dried on the filter under vacuum. Thesolid is dried to constant weight (<1% difference) in a vacuum oven at60° C. with a slight N₂ bleed to afford Compound 1 in Form I as anoff-white solid.

An X-ray diffraction pattern calculated from a single crystal structureof Compound 1 in Form I is shown in FIG. 1. Table 1 lists the calculatedpeaks for FIG. 1.

TABLE 1 Peak 2θ Angle Relative Intensity Rank [degrees] [%] 11 14.4148.2 8 14.64 58.8 1 15.23 100.0 2 16.11 94.7 3 17.67 81.9 7 19.32 61.3 421.67 76.5 5 23.40 68.7 9 23.99 50.8 6 26.10 67.4 10 28.54 50.1

An actual X-ray powder diffraction pattern of Compound 1 in Form I isshown in FIG. 2. Table 2 lists the actual peaks for FIG. 2.

TABLE 2 Peak 2θ Angle Relative Intensity Rank [degrees] [%] 7 7.83 37.73 14.51 74.9 4 14.78 73.5 1 15.39 100.0 2 16.26 75.6 6 16.62 42.6 517.81 70.9 9 21.59 36.6 10 23.32 34.8 11 24.93 26.4 8 25.99 36.9

An overlay of an X-ray diffraction pattern calculated from a singlecrystal structure of Compound 1 in Form I, and an actual X-ray powderdiffraction pattern of Compound 1 in Form I is shown in FIG. 3. Theoverlay shows good agreement between the calculated and actual peakpositions, the difference being only about 0.15 degrees.

The DSC trace of Compound 1 in Form I is shown in FIG. 4. Melting forCompound 1 in Form I occurs at about 204° C.

Conformational pictures of Compound 1 in Form I based on single crystalX-ray analysis are shown in FIGS. 5-8. FIGS. 6-8 show hydrogen bondingbetween carboxylic acid groups of a dimer and the resulting stackingthat occurs in the crystal. The crystal structure reveals a densepacking of the molecules. Compound 1 in Form I is monoclinic, P2₁/n,with the following unit cell dimensions: a=4.9626(7) Å, b=12.299(2) Å,c=33.075 (4) Å, 13=93.938(9)°, V=2014.0 Å³, Z=4. Density of Compound 1in Form I calculated from structural data is 1.492 g/cm³ at 100 K.

¹HNMR spectra of Compound 1 are shown in FIGS. 9-11 (FIGS. 9 and 10depict Compound 1 in Form I in a 50 mg/mL, 0.5 methylcellulose-polysorbate 80 suspension, and FIG. 11 depicts Compound 1 asan HCl salt).

Table 3 below recites additional analytical data for Compound 1.

TABLE 3 Cmpd. LC/MS LC/RT No. M + 1 min NMR 1 453.3 1.93 H NMR (400 MHz,DMSO-d6) 9.14 (s, 1H), 7.99-7.93 (m, 3H), 7.80-7.78 (m, 1H), 7.74-7.72(m, 1H), 7.60-7.55 (m, 2H), 7.41-7.33 (m, 2H), 2.24 (s, 3H), 1.53-1.51(m, 2H), 1.19-1.17 (m, 2H)

Assays

Assays for Detecting and Measuring ΔF508-CFTR Correction Properties ofCompounds

Membrane Potential Optical Methods for Assaying ΔF508-CFTR ModulationProperties of Compounds

The optical membrane potential assay utilized voltage-sensitive FRETsensors described by Gonzalez and Tsien (See Gonzalez, J. E. and R. Y.Tsien (1995) “Voltage sensing by fluorescence resonance energy transferin single cells” Biophys J 69(4): 1272-80, and Gonzalez, J. E. and R. Y.Tsien (1997) “Improved indicators of cell membrane potential that usefluorescence resonance energy transfer” Chem Biol 4(4): 269-77) incombination with instrumentation for measuring fluorescence changes suchas the Voltage/Ion Probe Reader (VIPR) (See, Gonzalez, J. E., K. Oades,et al. (1999) “Cell-based assays and instrumentation for screeningion-channel targets” Drug Discov Today 4(9): 431-439).

These voltage sensitive assays are based on the change in fluorescenceresonant energy transfer (FRET) between the membrane-soluble,voltage-sensitive dye, DiSBAC₂(3), and a fluorescent phospholipid,CC2-DMPE, which is attached to the outer leaflet of the plasma membraneand acts as a FRET donor. Changes in membrane potential (V_(m)) causethe negatively charged DiSBAC₂(3) to redistribute across the plasmamembrane and the amount of energy transfer from CC2-DMPE changesaccordingly. The changes in fluorescence emission were monitored usingVIPR™ II, which is an integrated liquid handler and fluorescent detectordesigned to conduct cell-based screens in 96- or 384-well microtiterplates.

1. Identification of Correction Compounds

To identify small molecules that correct the trafficking defectassociated with ΔF508-CFTR; a single-addition HTS assay format wasdeveloped. The cells were incubated in serum-free medium for 16 hrs at37° C. in the presence or absence (negative control) of test compound.As a positive control, cells plated in 384-well plates were incubatedfor 16 hrs at 27° C. to “temperature-correct” ΔF508-CFTR. The cells weresubsequently rinsed 3× with Krebs Ringers solution and loaded with thevoltage-sensitive dyes. To activate ΔF508-CFTR, 10 μM forskolin and theCFTR potentiator, genistein (20 μM), were added along with CL-freemedium to each well. The addition of CL-free medium promoted Cl⁻ effluxin response to ΔF508-CFTR activation and the resulting membranedepolarization was optically monitored using the FRET-basedvoltage-sensor dyes.

2. Identification of Potentiator Compounds

To identify potentiators of ΔF508-CFTR, a double-addition HTS assayformat was developed. During the first addition, a CL-free medium withor without test compound was added to each well. After 22 sec, a secondaddition of Cl⁻-free medium containing 2-10 μM forskolin was added toactivate ΔF508-CFTR. The extracellular Cl⁻ concentration following bothadditions was 28 mM, which promoted Cl⁻ efflux in response to ΔF508-CFTRactivation and the resulting membrane depolarization was opticallymonitored using the FRET-based voltage-sensor dyes.

3. Solutions Bath Solution #1: (in mM) NaCl 160, KCl 4.5, CaCl₂ 2, MgCl₂1, HEPES 10, pH 7.4 with NaOH. Chloride-free bath solution: Chloridesalts in Bath Solution #1 are substituted with gluconate salts.CC2-DMPE: Prepared as a 10 mM stock solution in DMSO and stored at −20°C. DiSBAC₂(3): Prepared as a 10 mM stock in DMSO and stored at −20° C.

4. Cell Culture

NIH3T3 mouse fibroblasts stably expressing ΔF508-CFTR are used foroptical measurements of membrane potential. The cells are maintained at37° C. in 5% CO₂ and 90% humidity in Dulbecco's modified Eagle's mediumsupplemented with 2 mM glutamine, 10% fetal bovine serum, 1×NEAA, β-ME,1× pen/strep, and 25 mM HEPES in 175 cm² culture flasks. For all opticalassays, the cells were seeded at 30,000/well in 384-well matrigel-coatedplates and cultured for 2 hrs at 37° C. before culturing at 27° C. for24 hrs for the potentiator assay. For the correction assays, the cellsare cultured at 27° C. or 37° C. with and without compounds for 16-24hours.

Electrophysiological Assays for Assaying ΔF508-CFTR ModulationProperties of Compounds

1. Using Chamber Assay

Using chamber experiments were performed on polarized epithelial cellsexpressing ΔF508-CFTR to further characterize the ΔF508-CFTR modulatorsidentified in the optical assays. FRT^(ΔF508-CFTR) epithelial cellsgrown on Costar Snapwell cell culture inserts were mounted in an Ussingchamber (Physiologic Instruments, Inc., San Diego, Calif.), and themonolayers were continuously short-circuited using a Voltage-clampSystem (Department of Bioengineering, University of Iowa, Iowa, and,Physiologic Instruments, Inc., San Diego, Calif.). Transepithelialresistance was measured by applying a 2-mV pulse. Under theseconditions, the FRT epithelia demonstrated resistances of 4 KΩ/cm² ormore. The solutions were maintained at 27° C. and bubbled with air. Theelectrode offset potential and fluid resistance were corrected using acell-free insert. Under these conditions, the current reflects the flowof Cl⁻ through ΔF508-CFTR expressed in the apical membrane. The I_(SC)was digitally acquired using an MP100A-CE interface and AcqKnowledgesoftware (v3.2.6; BIOPAC Systems, Santa Barbara, Calif.).

2. Identification of Correction Compounds

Typical protocol utilized a basolateral to apical membrane Cl⁻concentration gradient. To set up this gradient, normal ringer was usedon the basolateral membrane, whereas apical NaCl was replaced byequimolar sodium gluconate (titrated to pH 7.4 with NaOH) to give alarge Cl⁻ concentration gradient across the epithelium. All experimentswere performed with intact monolayers. To fully activate ΔF508-CFTR,forskolin (10 μM) and the PDE inhibitor, IBMX (100 μM), were appliedfollowed by the addition of the CFTR potentiator, genistein (50 μM).

As observed in other cell types, incubation at low temperatures of FRTcells stably expressing ΔF508-CFTR increases the functional density ofCFTR in the plasma membrane. To determine the activity of correctioncompounds, the cells were incubated with 10 μM of the test compound for24 hours at 37° C. and were subsequently washed 3× prior to recording.The cAMP- and genistein-mediated I_(SC) in compound-treated cells wasnormalized to the 27° C. and 37° C. controls and expressed as percentageactivity. Preincubation of the cells with the correction compoundsignificantly increased the cAMP- and genistein-mediated I_(SC) comparedto the 37° C. controls.

3. Identification of Potentiator Compounds

Typical protocol utilized a basolateral to apical membrane Cl⁻concentration gradient. To set up this gradient, normal ringers was usedon the basolateral membrane and was permeabilized with nystatin (360μg/ml), whereas apical NaCl was replaced by equimolar sodium gluconate(titrated to pH 7.4 with NaOH) to give a large Cl⁻ concentrationgradient across the epithelium. All experiments were performed 30 minafter nystatin permeabilization. Forskolin (10 μM) and all testcompounds were added to both sides of the cell culture inserts. Theefficacy of the putative ΔF508-CFTR potentiators was compared to that ofthe known potentiator, genistein.

4. Solutions Basolateral NaCl (135), CaCl₂ (1.2), MgCl₂ (1.2), K₂HPO₄solution (in (2.4), KHPO₄ (0.6), N-2-hydroxyethylpiperazine- mM):N′-2-ethanesulfonic acid (HEPES) (10), and dextrose (10). The solutionwas titrated to pH 7.4 with NaOH. Apical solution Same as basolateralsolution with NaCl replaced (in mM): with Na Gluconate (135).

5. Cell Culture

Fisher rat epithelial (FRT) cells expressing ΔF508-CFTR(FRT^(ΔF508-CFTR)) were used for Ussing chamber experiments for theputative ΔF508-CFTR modulators identified from our optical assays. Thecells were cultured on Costar Snapwell cell culture inserts and culturedfor five days at 37° C. and 5% CO₂ in Coon's modified Ham's F-12 mediumsupplemented with 5% fetal calf serum, 100 U/ml penicillin, and 100μg/ml streptomycin. Prior to use for characterizing the potentiatoractivity of compounds, the cells were incubated at 27° C. for 16-48 hrsto correct for the ΔF508-CFTR. To determine the activity of correctionscompounds, the cells were incubated at 27° C. or 37° C. with and withoutthe compounds for 24 hours.

6. Whole-Cell Recordings

The macroscopic ΔF508-CFTR current (I_(ΔF508)) in temperature- and testcompound-corrected NIH3T3 cells stably expressing ΔF508-CFTR weremonitored using the perforated-patch, whole-cell recording. Briefly,voltage-clamp recordings of I_(ΔF508) were performed at room temperatureusing an Axopatch 200B patch-clamp amplifier (Axon Instruments Inc.,Foster City, Calif.). All recordings were acquired at a samplingfrequency of 10 kHz and low-pass filtered at 1 kHz. Pipettes had aresistance of 5-6 MΩ when filled with the intracellular solution. Underthese recording conditions, the calculated reversal potential for Cl⁻(E_(Cl)) at room temperature was −28 mV. All recordings had a sealresistance >20 GΩ and a series resistance <15 MΩ. Pulse generation, dataacquisition, and analysis were performed using a PC equipped with aDigidata 1320 A/D interface in conjunction with Clampex 8 (AxonInstruments Inc.). The bath contained <250 μl of saline and wascontinuously perifused at a rate of 2 ml/min using a gravity-drivenperfusion system.

7. Identification of Correction Compounds

To determine the activity of correction compounds for increasing thedensity of functional ΔF508-CFTR in the plasma membrane, we used theabove-described perforated-patch-recording techniques to measure thecurrent density following 24-hr treatment with the correction compounds.To fully activate ΔF508-CFTR, 10 μM forskolin and 20 μM genistein wereadded to the cells. Under our recording conditions, the current densityfollowing 24-hr incubation at 27° C. was higher than that observedfollowing 24-hr incubation at 37° C. These results are consistent withthe known effects of low-temperature incubation on the density ofΔF508-CFTR in the plasma membrane. To determine the effects ofcorrection compounds on CFTR current density, the cells were incubatedwith 10 μM of the test compound for 24 hours at 37° C. and the currentdensity was compared to the 27° C. and 37° C. controls (% activity).Prior to recording, the cells were washed 3× with extracellularrecording medium to remove any remaining test compound. Preincubationwith 10 μM of correction compounds significantly increased the cAMP- andgenistein-dependent current compared to the 37° C. controls.

8. Identification of Potentiator Compounds

The ability of ΔF508-CFTR potentiators to increase the macroscopicΔF508-CFTR Cl⁻ current (I_(ΔF508)) in NIH3T3 cells stably expressingΔF508-CFTR was also investigated using perforated-patch-recordingtechniques. The potentiators identified from the optical assays evoked adose-dependent increase in I_(ΔF508) with similar potency and efficacyobserved in the optical assays. In all cells examined, the reversalpotential before and during potentiator application was around −30 mV,which is the calculated E_(Cl) (−28 mV).

9. Solutions Intracellular Cs-aspartate (90), CsCl (50), MgCl₂ (1),HEPES solution (in (10), and 240 μg/ml amphotericin-B (pH mM): adjustedto 7.35 with CsOH). Extracellular N-methyl-D-glucamine (NMDG)-Cl (150),solution (in MgCl₂ (2), CaCl₂ (2), HEPES (10) (pH adjusted mM): to 7.35with HCl).

10. Cell Culture

NIH3T3 mouse fibroblasts stably expressing ΔF508-CFTR are used forwhole-cell recordings. The cells are maintained at 37° C. in 5% CO₂ and90% humidity in Dulbecco's modified Eagle's medium supplemented with 2mM glutamine, 10% fetal bovine serum, 1×NEAA, β-ME, 1× pen/strep, and 25mM HEPES in 175 cm² culture flasks. For whole-cell recordings,2,500-5,000 cells were seeded on poly-L-lysine-coated glass coverslipsand cultured for 24-48 hrs at 27° C. before use to test the activity ofpotentiators; and incubated with or without the correction compound at37° C. for measuring the activity of correctors.

11. Single-Channel Recordings

The single-channel activities of temperature-corrected ΔF508-CFTR stablyexpressed in NIH3T3 cells and activities of potentiator compounds wereobserved using excised inside-out membrane patch. Briefly, voltage-clamprecordings of single-channel activity were performed at room temperaturewith an Axopatch 200B patch-clamp amplifier (Axon Instruments Inc.). Allrecordings were acquired at a sampling frequency of 10 kHz and low-passfiltered at 400 Hz. Patch pipettes were fabricated from Corning KovarSealing #7052 glass (World Precision Instruments, Inc., Sarasota, Fla.)and had a resistance of 5-8 MΩ when filled with the extracellularsolution. The ΔF508-CFTR was activated after excision, by adding 1 mMMg-ATP, and 75 nM of the cAMP-dependent protein kinase, catalyticsubunit (PKA; Promega Corp. Madison, Wis.). After channel activitystabilized, the patch was perifused using a gravity-drivenmicroperfusion system. The inflow was placed adjacent to the patch,resulting in complete solution exchange within 1-2 sec. To maintainΔF508-CFTR activity during the rapid perfusion, the nonspecificphosphatase inhibitor F (10 mM NaF) was added to the bath solution.Under these recording conditions, channel activity remained constantthroughout the duration of the patch recording (up to 60 min). Currentsproduced by positive charge moving from the intra- to extracellularsolutions (anions moving in the opposite direction) are shown aspositive currents. The pipette potential (V_(p)) was maintained at 80mV.

Channel activity was analyzed from membrane patches containing ≦2 activechannels. The maximum number of simultaneous openings determined thenumber of active channels during the course of an experiment. Todetermine the single-channel current amplitude, the data recorded from120 sec of ΔF508-CFTR activity was filtered “off-line” at 100 Hz andthen used to construct all-point amplitude histograms that were fittedwith multigaussian functions using Bio-Patch Analysis software(Bio-Logic Comp. France). The total microscopic current and openprobability (P_(o)) were determined from 120 sec of channel activity.The P_(o) was determined using the Bio-Patch software or from therelationship P_(o)=I/i(N), where I=mean current, i=single-channelcurrent amplitude, and N=number of active channels in patch.

12. Solutions Extracellular NMDG (150), aspartic acid (150), CaCl₂ (5),solution (in mM): MgCl₂ (2), and HEPES (10) (pH adjusted to 7.35 withTris base). Intracellular NMDG-Cl (150), MgCl₂ (2), EGTA (5), TESsolution (in mM): (10), and Tris base (14) (pH adjusted to 7.35 withHCl).

13. Cell Culture

NIH3T3 mouse fibroblasts stably expressing ΔF508-CFTR are used forexcised-membrane patch-clamp recordings. The cells are maintained at 37°C. in 5% CO₂ and 90% humidity in Dulbecco's modified Eagle's mediumsupplemented with 2 mM glutamine, 10% fetal bovine serum, 1×NEAA, β-ME,1× pen/strep, and 25 mM HEPES in 175 cm² culture flasks. For singlechannel recordings, 2,500-5,000 cells were seeded onpoly-L-lysine-coated glass coverslips and cultured for 24-48 hrs at 27°C. before use.

Using the procedures described above, the activity, i.e., EC50s, ofCompound 1 has been measured and is shown in Table 4.

TABLE 4 IC50/EC50 Bins: +++ <= 2.0 < ++ <= 5.0 < + PercentActivityBins: + <= 25.0 < ++ <= 100.0 < +++ Cmpd. No. BinnedEC50BinnedMaxEfficacy 1 +++ +++

We claim:
 1. A process for preparing3-(6-(1-(2,2-Difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoicacid characterized as Form I, comprising suspending or dissolving theHCl salt of3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoicacid in an appropriate solvent for an effective amount of time.
 2. Theprocess of claim 1, wherein the appropriate solvent is water or 50%methanol/water mixture.
 3. The process of claim 1, wherein theappropriate solvent is water.
 4. The process of claim 1, wherein theeffective amount of time is 2 to 24 hours.
 5. The process of claim 1,wherein the effective amount of time is 2 to 6 hours.